This invention relates to an alkaline lipolytic enzyme, a detergent composition comprising the enzyme, methods of producing the enzyme, an isolated DNA sequence encoding the enzyme, a recombinant expression vector comprising the DNA sequence and cells comprising the DNA sequence or the vector.
For a number of years lipolytic enzymes have been used as detergent additives to remove lipid or fatty stains.
Thus, the prior art suggests the use of various lipolytic enzymes with lipase or cutinase activity as detergent additives. Examples include microbial lipolytic enzymes derived from strains of Fusarium, e.g. F. oxysporum (EP 130 064) and F. solani f. sp. pisi (WO 90/09446), Humicola lanuginosa (also called Thermomyces lanuginosus, EP 258 068 and EP 305 216), Pseudomonas, e.g. P. alcaligenes and P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. mendocina (WO 88/09367), and Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260), B. stearothermophilus (JP 64/74992) and B. pumilus (WO 91/16422).
It is the object of this invention to provide lipolytic enzymes having good washing performance and stability in a detergent solution.
Surprisingly, we have found that alkaline lipolytic enzymes can be obtained from filamentous fungi of the genera Gliocladium, Verticillium and Trichophaea and that the lipolytic enzymes are effective for improving the effect of detergents. The lipolytic enzymes have a good washing performance and stability in a detergent solution.
Full length cDNA sequences each encoding a lipolytic enzyme according to the invention were derived from three strains of Giiocladium sp., Verticillium sp. and Trichophaea saccata as donor organisms. The cDNA sequences were cloned into the plasmid pYES 2.0 present in Escherichia coli., and the cloned E. coli strains were deposited by the inventors, as shown in the table below. The lipolytic enzyme encoding DNA sequence harbored in the deposited E. coli strain is believed to have the sequence shown in the positions and listing indicated below, and the amino acid sequence deduced therefrom is shown in the indicated positions and listing.
The information is summarized below:
Homologies of the above DNA and amino acid sequences were calculated by methods described later in this specification. The following homologies were found between pairs of sequences, amino acid homology at the upper right corner, and DNA homology at the lower left. (given as DNA homology/amino acid homology):
Accordingly, the invention in its various aspects provides:
1. A lipolytic enzyme which is:
a) a polypeptide encoded by the lipolytic enzyme encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli DSM 10591, DSM 10590 or DSM 11298, or
b) a polypeptide produced by Gliocladium sp. CBS 173.96, Verticillium sp. CBS 830.95 or Trichophaea saccata CBS 804.70, or
c) a polypeptide having an amino acid sequence as shown in positions 1-200 of SEQ ID NO: 3, positions 1-202 of SEQ ID NO: 6, or positions 1-201 of SEQ ID NO: 8, or
d) an analogue of the polypeptide defined in (a), (b) or (c) which:
i) is at least 60% homologous with said polypeptide, or
ii) is immunologically reactive with an antibody raised against said polypeptide in purified form.
2. An alkaline lipolytic enzyme which is derivable from a strain of Gliocladium and has a lipolytic activity at pH 10 in the absence of Ca++ above 20% of the lipolytic activity at pH 10 in the presence of 50 mM Ca++.
3. An alkaline lipolytic enzyme which is derivable from a strain of Gliocladium and gives a degree of hydrolysis above 15% on cotton/olive oil swatches in the Activity-in-Detergent (AiD) assay.
4. An alkaline lipolytic enzyme which is derivable from a strain of the genus Verticillium and retains more than 90% activity after 30 minutes incubation at pH 10.2, 40xc2x0 C. in a solution of 0.300 g/l C14-C16 alkyl sulfate, 0.650 g/l alcohol ethoxylate (C12-C14, 6 EO), 1.750 zeolite P, 0.145 g/l Na2CO3, 0.020 g/l acrylate/maleate copolymer and 0.050 g/l carboxymethyl cellulose.
5. An enzymatic detergent composition comprising a surfactant and the lipolytic enzyme of any preceding claim.
6. A method of producing an alkaline lipolytic enzyme, comprising cultivation of a lipolytic enzyme-producing strain of Gliocladium, Verticillium or Trichophaea in a suitable nutrient medium, followed by recovery of the alkaline lipolytic enzyme.
7. A method for producing an alkaline lipolytic enzyme, comprising:
a) isolating a DNA sequence encoding the lipolytic enzyme from a lipolytic enzyme-producing strain of Gliocladium, Verticillium or Trichophaea,
b) combining the DNA fragment with appropriate expression signal(s) in an appropriate vector,
c) transforming a suitable heterologous host organism with the vector,
d) cultivating the transformed host organism under conditions leading to expression of the lipolytic enzyme, and
e) recovering the lipolytic enzyme from the culture medium.
8. An isolated DNA sequence which encodes the lipolytic enzyme of any of claims 1-7.
9. An isolated, lipolytic enzyme encoding DNA sequence which comprises:
a) the lipolytic enzyme encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli DSM 10591, DSM 10590 or DSM 11298, or
b) the DNA sequence shown in positions 114-713 of SEQ ID NO: 2, positions 133-738 of SEQ ID NO: 5 or positions 161-763 of SEQ ID NO: 7, or
c) an analogue of the DNA sequence defined in a) or b) which
i) is at least 60% homologous with said DNA sequence, or
ii) hybridizes with said DNA sequence at 55xc2x0 C.
10. A recombinant expression vector comprising the DNA sequence of any of claims 19-24.
11. A cell comprising the DNA sequence of any of claims 19-24 or the recombinant expression vector of claim 25.
12. A method of producing a lipolytic enzyme, comprising culturing the cell of any of claims 26-29 under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
13. A biologically pure culture of a microbial strain which belongs to the genus Gliocladium or Verticillium and is capable of producing an alkaline lipolytic enzyme.
14. Escherichia coli strain DSM 10591, DSM 10590 or DSM 11298 or a mutant thereof having lipolytic enzyme encoding capability.
Tilburg and Thomas, Application. Environ. Microbiol., January 1993, p. 236-242 describes production of lipase by G. virens; however, data in the article show that the prior-art lipase is not alkaline. U.S. Pat. Nos. 4,985,365 and 4,511,655 describe the use of culture broth of G. roseum IFO 5422 and G. virens IFO 6355 to hydrolyze carboxylic esters at acid pH. The prior art does not describe the production of lipolytic activity at alkaline pH by strains of Gliocladium.
The prior art describes the production of lipase by Verticillium cinnabarinum (also called V. Iuteoalbum) DSM 63078 (Rapp and Backhaus, Enzyme Microb. Technol., 14, 938-943 (1992)) and Verticillium lecanii ATCC 26854 (JP-A 61-289884). The inventors have investigated the two strains and found that they do not produce alkaline lipolytic enzyme.
The following literature describes lipase production by the genus Verticillium without identifying any particular strains: Kunert and Lysek, Biologica (Bratislava), 42 (3), 285-293 (1987). Leger et al., J. Invertebr. Pathol., 48, 85-95 (1986). Jackson et al., Ann. appl. Biol., 106, 39-48 (1985). Roberts et al., Mycologia, 79 (2), 265-273 (1987). Trigiano, Mycologia, 71, 908-917 (1979). However, the prior art does not describe the production of lipolytic activity at alkaline pH by strains of Verticillium.
A homology search was performed in nucleotide and protein databases. The highest homology for the lipolytic enzyme and DNA sequences of the invention was found with the sequence for cutinase from Fusarium solani f. sp. pisi, described by C. L. Soliday et al., Proc. Natl. Acad. Sci. USA, 81, 3939-3943 (1984).
The three DNA sequences of the invention described earlier in this specification show homologies of 53-57% with the above known DNA sequence, and the three amino acid sequences of the invention described earlier show homologies of 50-53% with the above known amino acid sequence. The calculation of homology was done as described later in this specification. Using a formula given in xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d, John Wiley and Sons, 1995, hybridization of the above DNA of the invention and the closest prior-art DNA is estimated to have a melting temperature of 50xc2x0 C. at the hybridization conditions given later in this specification.